The human metapneumovirus (hMPV, hereinafter) is the etiologic agent of a high percentage of hospitalizations and morbidity associated with acute respiratory infections of the upper and lower respiratory tracts, especially in infants, elderly and immunocompromised individuals. Infection with this virus is associated with a wide range of conditions, being bronchiolitis and pneumonia the conditions with a higher socio-economic impact. Additionally, it have been associated with gastroenteritis, and keratoconjunctivitis. Calvo et al. (2008) demonstrated in a 3 years period study that cumulative incidence of acute respiratory infections caused by respiratory viruses RSV, ADV and hMPV accounted for 64.5% of hospital admissions of children younger than 2 years, being the incidence for each virus 35.4%, 19.3% and 9.8%, respectively. One interesting feature that hMPV shares with the other high incidence respiratory viruses is the production of repeated infections throughout childhood, a phenomenon possibly associated with a failure in the establishment of a protective immune response to the first infection during early months of life. This latest phenomenon motivates the urgent need for public health systems to have new prototype of vaccines with the ability to control annual outbreaks of respiratory infections, thereby allowing relieve congestion in healthcare institutions and ultimately the socio-economic impact associated with these infections. To date, there are no studies about the specific economic impact of hMPV infection, however, the incidence of hospitalization for hMPV has been estimated in ⅓ of the incidence of hospitalization for human respiratory syncytial virus (hRSV). Studies conducted in developed countries estimate individual cost of hRSV infection about 3,000 euros ($1.86 million Chilean pesos) with an upper limit of up to 8,400 euros ($5.2 million Chilean pesos). The costs associated to individual hospitalization are approximate and based on a pathological process of similar characteristics that requires hospitalization.
The hMPV virus is classified in the family Paramyxoviridae subfamily Pneumovirinae, the same family in which hRSV is classified, although each one is grouped within the Metapneumovirus and Pneumovirus genus, respectively. HMPV genome comprises a non-segmented, single-stranded, negative-sense ribonucleic acid (ssRNA), so that the viral proteins are arranged in a 3′ to 5′ direction (with respect to their sequence) as follows: N, P, M, F, M2 (ORF1 and 2), SH, G and L. Five of these proteins are responsible for packaging the genetic material and defining the structure of the viral particle, corresponding to the nucleocapsid protein N and the matrix protein M, together with transmembrane glycoproteins F, G and SH, respectively. The other four proteins, M2-1, M2-2, P and L, are involved in viral replication and transcription. There are two subtypes of hMPV, classified as A and B relative to two antigenic groups based on sequence differences primarily in proteins F and G. Although these proteins have some degree of difference, there is a high identity compared to other proteins encoded by the viral genome. The development of vaccines against respiratory viruses began in the 1960s with the first prototype of hRSV vaccine based on formalin-inactivated virus (hRSV-FI), which had significant adverse effects that prevented its use in immunization programs. The intramuscular formulation together with aluminum hydroxide adjuvant produced in vaccinated infants more severe symptoms than those in infected individuals not vaccinated. This effect was associated with a hyper-responsiveness of the immune response to infection, characterized by a large parenchymal infiltration of polymorphonuclear cells, eosinophils and neutrophils and a high titer of complement-fixing antibodies.
For human metapneumovirus (hMPV) only few vaccines have been developed and so far none has had a satisfactory result. A prototype using the same previous design of formalin-inactivated virus (hMPV-FI), also produced inflammatory hyper-responsiveness symptoms with similar characteristics to that produced by the vaccine against hRSV in a Sigmodon hispidus infection model (Yim et al., 2007). In contrast with the hyper-responsiveness processes observed for hSRV, partial elimination of the virus from the respiratory system was demonstrated. The disease observed in mice and Sigmodon hispidus exposed to hSRV-Fl was associated with an immunopathological response based on Th2-type antibodies and an exaggerated activation of NF-κB. Increased NF-κB transcriptional activity further relates to the secretion of pro-inflammatory cytokines such as IL-8. Furthermore, the hyper-responsiveness of lung tissue after hMPV infection has been associated with immune responses characterized by presence of IFN-γ and IL-4 in bronchoalveolar lavage and detection of IgG1 and IgG2a neutralizing antibodies in sick mice serum, strongly suggesting that chronic inflammation observed is due either to pathological responses of Th1 and Th2 type or an insufficient response based on Th1-type cells accompanied by a pathological Th2 response. As for the latter assumption, some authors have proposed that an increased Th1 response could also exacerbate the disease.
These facts emphasize the importance of establishing a balanced and efficient immune response able to limit the progress of the inflammatory process and, in turn, induce the proper clearance of these viruses from infected tissues.
Because the respiratory disease caused by hMPV is similar to what was previously observed with hSRV, which has been associated with a failure in the induction of cellular immunity, it is necessary to generate a prototype vaccine which is a good inducer of CD8+ cytotoxic T and CD4+ helper T cells, both IFN-γ producers. Recent experimental approaches have focused their efforts on developing vaccines only towards one viral species, using different techniques of molecular genetics and immunology. It is important to mention that these studies have used a limited number of proteins or protein subunits as antigens for each virus of interest. For hSRV, some of them have been based on the use of individual viral proteins, such as whole subunits or fragments of F or G proteins, or a mixture thereof in murine and non-human primate models of infection. Some vaccine prototypes against hSRV have been used in phase I and II clinical trials, but the results have not shown a long-term protective ability, and vaccines are far from suitable for extensive use in the prophylaxis of infection (Denis et al., 2005; Karron et al., 2005).
Attenuated hMPV strains have been developed by eliminating genes that have been suggested are related to viral pathogenicity. HMPV strains lacking of genes encoding for SH, G, and, M2-1 and M2-2 proteins have been proposed as vaccine candidates (Biacchesi et al., 2005). These candidates have showed good results in animal models but have not yet been studied in humans. Although this alternative is viable, it is very expensive as it requires the production of virus in cell cultures approved for human use. Another source of vaccine is the overexpression of viral proteins by heterologous systems, such as hMPV F-protein coupled with adjuvants for generate neutralizing antibodies, but the disadvantage of this option is that provides immunity for a very short period of time (Herfst et al., 2008a).
There are no clinical studies of vaccine candidates against hMPV, because the development of prototypes capable of generating protective immunity has failed. To date, prototypes evaluated in animal models generate Th2-type antibody-based immunity, which are not long-term or effective preventing infection. Animals vaccinated with these prototypes generate neutralizing antibodies in vitro, but they are not protected against infection or the clinical symptoms of disease induced by hMPV infection in mice (Cseke et al., 2007) or macaques (Herfst et al., 2008b) are reduced. The antibody-based immunity is not efficient neutralizing virus in vivo. Antibodies capable of neutralizing hMPV in vitro, are not able to prevent infection or disease caused by hMPV, when used as therapy via passive immunization (Hamelin et al., 2008). Moreover, it has been observed that the resolution of viral symptoms requires the participation of Th1-type cell-based immune response. It was shown that the resolution of the viral condition and clearance of viral particles is dependent on the activation of CD4+ and CD8+ lymphocytes, even though the activity of these is also responsible for the immunopathology of disease (Kolli et al., 2008). More recently it has been found that the primary effectors of viral symptoms resolution are CD8+-type cytotoxic cells, although CD4+ helper T lymphocytes have a regulatory involvement. Thus, a balanced immune response of Th1-type cells is needed to produce the resolution of viral symptoms without causing inflammatory hyper-responsiveness. In summary, currently there is no vaccine against human metapneumovirus (hMPV) able to give effective protection and with no major side effects, in fact, there are no commercial vaccines available against this virus.
Surprisingly the inventors have found that the use of a recombinant Mycobacterium strain expressing P-protein from human Metapneumovirus allows to generate a protective immunity against infection produced by respiratory Metapneumovirus virus without causing unacceptable side effects, such as inflammatory hyper-responsiveness in airways. This invention solves a technical problem that remained unsolved in the prior art, consisting of an immunogenic formulation that provides protection against infections by hMPV and not generate inflammatory hyper-responsiveness.